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Leveraging the Power of in Vitro Transcription: an Exploration of Applications, Challenges and Solutions

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Leveraging the Power of in Vitro Transcription: an Exploration of Applications, Challenges and Solutions Leveraging the Power of In Vitro Transcription: An Exploration of Applications, Challenges and Solutions Rob Brazas, Ph.D. Senior Product Manager, Lucigen September, 2017 www.lucigen.com Agenda Leveraging In Vitro Transcription for Your Success • Overview of in vitro transcription (IVT) technology • Applications of IVT-produced RNA – Overview of each type of application – Examples of IVT use for each application with data from the literature • Key challenges of IVT and working with RNA • Solutions to those challenges – AmpliScribe™ T7-Flash Transcription Kit – DuraScribe® T7 Transcription Kit In Vitro Transcription (IVT) Transcription Generally Starts with a dsDNA Template In Vitro Transcription: Production of RNA in a tube (e.g. microfuge tube) using an RNA polymerase (usually phage derived), ribonucleotides and appropriate buffer conditions. DNA of interest (e.g. ORF, shRNA, aptamer) dsDNA 5’ 3’ Template 3’ T7 Promoter 5’ SP6 Promoter T3 Promoter IVT Template Options Multiple Forms of DNA Make Acceptable IVT Templates Blunt End 5’ 3’ Linearized Plasmid 3’ T7p 5’ 5’ 3’ Linearized Plasmid 3’ T7p 5’ 5’ Overhang 5’ 3’ PCR Product 3’ T7p 5’ +1 5’ 3’ 5’ TAATACGACTCACTATAG Synthetic Construct 3’ T7p 5’ 3’ ATTATGCTGAGTGATATCCCNNNNNNNNNNNNN 5’ T7 Promoter 2nd Strand cDNA 5’ AAAA 3’ cDNA 3’ TTTT T7p 5’ 1st Strand cDNA Caution: 3’ overhangs may lead to the production of spurious RNA transcripts IVT Reactions Production of Large Amounts of RNA from ~1 µg of DNA T7 RNA Polymerase + ATP, UTP, GTP & CTP, reaction buffer, +/- RNase Inhibitor DNA of interest (e.g. ORF, shRNA, aptamer) dsDNA 5’ 3’ Template 3’ T7 Promoter 5’ T7 RNA Polymerase 5’ 3’ 3’ T7 Promoter 5’ T7 RNA Polymerase 5’ T7 RNA 5’ Polymerase 3’ 3’ T7 Promoter 5’ IVT RNA transcripts 5’ 3’ Production of Modified or Labeled IVT RNA Modified or Labeled NTPs are Effectively Incorporated + Reaction buffer T7 RNA +/- RNase Inhibitor Polymerase + ATP, UTP, GTP & CTP + Labeled or modified ribonucleotides (e.g. Biotin-UTP, Digoxigenin-UTP, 2’-F-UTP*) 5’ 3’ 3’ T7 Promoter 5’ Biotin Biotin Biotin Biotin-RNA U U U U U Dig Dig Dig Digoxigenin-RNA U U U U U 2’-F-RNA* U UF U UF UF * Incorporation of 2’-F-UTP and 2’-F-CTP requires a mutated T7 RNA Polymerase Note: It is also possible to incorporate radiolabeled ribonucleotides Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible Viral RNA synthesis and transfection to start replication mRNA synthesis for in vitro and in vivo applications RNA structure studies RNA aptamer synthesis for SELEX dsRNA or shRNA synthesis for RNAi CRISPR gRNA synthesis for RNP-mediated gene editing Riboprobe synthesis for Northern blotting, in situ hybridization RNA amplification → amplified cDNA (MessageBOOSTER™ Kits) miRNA synthesis Anti-sense RNA synthesis More… you’re only limited by your imagination! Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible Viral RNA synthesis and transfection to start replication mRNA synthesis for in vitro and in vivo applications RNA structure studies RNA aptamer synthesis for SELEX dsRNA or shRNA synthesis for RNAi CRISPR gRNA synthesis for RNP-mediated gene editing Riboprobe synthesis for Northern blotting, in situ hybridization RNA amplification → amplified cDNA (MessageBOOSTER™ Kits) miRNA synthesis Anti-sense RNA synthesis More… you’re only limited by your imagination! Flaviviradae Family Viruses Great Models for All Things Related to IVT RNA Flaviviridae Example: Hepatitis C Virus IRES From: Moradpour, Penin and Rice (2007) Replication of Hepatitis C virus. Nature Reviews | Microbiology 5:453-463 Hepatitis C Virus • 9.6 kb +strand RNA genome – Flaviviridae family (e.g. Yellow fever, Dengue) • After cellular entry, cell translates genomic RNA into a polyprotein using IRES to initiate • Polyprotein is cleaved by cellular protease (diamonds) and viral proteases (arrows) o Proteolysis occurs co- and post-translationally Hepatitis C Virus Replication Cycle Replication Starts with Translation of the Incoming Genome From: Moradpour, Penin and Rice (2007) Replication of Hepatitis C virus. Nature Reviews | Microbiology 5:453-463 a) Cell binding and entry d) RNA genome replication (+RNA) → (-) RNA → (+) RNA b) Uncoating and genome release e) Packaging and assembly c) IRES-mediated translation and polyprotein processing f) Maturation and release Translation of IVT RNA Uncapped IVT RNAs are Poorly Translated T7 RNA Polymerase 5’ 3’ T7 Promoter ORF 5’ Poor translation, Uncapped mRNA unstable + Post-transcriptional PolyA Tailing Poor translation, Uncapped/PolyA+ mRNA A n improved stability Translation of IVT RNA Addition of a m7-G Cap Analog Improves Translation T7 RNA 7 7 Polymerase 5’-m G Cap Analog (m G(5')ppp(5')G) added to IVT reaction 5’ 3’ T7 Promoter ORF 5’ Improved translation, Capped mRNA m7-G unstable + Post-transcriptional PolyA Tailing Improved translation & Cap/Poly(A) mRNA m7-G A n stability Cap Analogs (ARCA, Normal) Note: 5’ Caps can also be added 3’ post-transcriptionally (OH) Translation of IVT RNA Viral IRES Enhance Translation in the Absence of a Cap IRES = Internal Ribosome Entry Site T7 RNA Polymerase 5’ 3’ 3’ T7 Promoter IRES ORF 5’ Improved IRES - mRNA 5’ 3’ translation Translation of IVT RNA IVT mRNA can be Translated In Vivo and In Vitro me-G An Cell-free protein expression Transfection into cultured cells • Rabbit reticulocyte extracts Microinjection (e.g. oocytes) • Wheat germ extracts • HeLa extracts Benefits Benefits • Speed • Transient/short term expression • No cellular toxicity • Pulse of expression • Better solubility • No potential DNA effects Applications Applications • Protein:protein interactions • RNA processing, stability studies • Structure studies • Protein expression • Enzyme/functional assays o e.g. CRISPR nucleases Transfection of YFV, HCV Viral RNAs Made In Vitro Transfected RNAs are Translated and Initiate Replication T7p YFV Replicon with Luciferase ORF T7p HCV Replicon with NeoR ORF In vitro transcription In vitro transcription 5’ 3’ 5’ 3’ Transfection, growth, Transfection, growth, dilution & luciferase assay replating, colony staining (-) control (+) control From: Gonzalez et. al. (2007) Selection of an optimal RNA transfection reagent and comparison to electroporation for the delivery of viral RNA. Journal of Virological Methods. 145:14-21 Using In Vitro Transcription and Translation to Identify HCV Protein: Host Protein Interactions by GST Pull-downs T7p HCV NS5B ORF In vitro transcription 5’ 3’ From: Domitrovic et. al. (2005) Role of La autoantigen and polypyrimidine tract-binding In vitro translation – RRL protein in HCV replication. Virology. 335:72-86 +35S-methionine B GST + NS5B Mix, bind, B GST La NS5B then wash + B GST PTB + NS5B B GST B GST La Electrophorese on SDS-PAGE 35 B GST PTB NS5B and image S-signals Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible Viral RNA synthesis and transfection to start replication mRNA synthesis for in vitro and in vivo applications RNA structure studies RNA aptamer synthesis for SELEX dsRNA or shRNA synthesis for RNAi CRISPR gRNA synthesis for RNP-mediated gene editing Riboprobe synthesis for Northern blotting, in situ hybridization RNA amplification → amplified cDNA (MessageBOOSTER™ Kits) miRNA synthesis Anti-sense RNA synthesis More… you’re only limited by your imagination! Using In Vitro Transcribed HCV IRES RNA to Determine Its Crystal Structure T7p HCV IRES Central Domain HDV Ribozyme In vitro transcription 5’ 3’ From: Berry et. al. (2011) Crystal Structure of the HDV Ribozyme cleavage HCV IRES Central Domain Reveals Strategy for Start- Codon Positioning. Cell. 19:1456-1466 5’ 3’ 5’ 3’ HCV IRES Central Core Cleanup, crystallization, crystallography Central Domain Structure Model of IRES on 40S Ribosomal Subunit Uses/Applications of IVT RNA IVT RNA Makes Many Experiments Possible Viral RNA synthesis and transfection to start replication mRNA synthesis for in vitro and in vivo applications RNA structure studies RNA aptamer synthesis for SELEX dsRNA or shRNA synthesis for RNAi CRISPR gRNA synthesis for RNP-mediated gene editing Riboprobe synthesis for Northern blotting, in situ hybridization RNA amplification → amplified cDNA (MessageBOOSTER™ Kits) miRNA synthesis Anti-sense RNA synthesis More… you’re only limited by your imagination! What Are RNA Aptamers? Think of Them as RNA-based Antibodies ssRNA Aptamer with Predicted Single-stranded RNA molecules Secondary Structure that: • Fold into RNA structure with secondary and tertiary structure • Generally 40 to 100 nucleotides long • Correct sequences/structures Folds into 3D structure that tightly and bind tightly and specifically to a specifically binds its target target molecule (e.g., protein, toxin, cell receptor, other RNAs…) Think “Antibody made of RNA”… How Are Good RNA Aptamers Made and Identified? SELEX Systematic Evolution of Ligands by EXponential Enrichment T7p A Random B Chemical synthesis of selected aptamer dsDNA Library Sequence library In vitro transcription Target Binding dsDNA PCR 8 – 15X 3’ 5’ Washing 3’ 5’ cDNA Reverse transcription Elution Identification of Aptamers that Bind HCV NS5B Replicase Used Both Standard RNA and 2’-F-CTP, -UTP Substituted RNA Standard RNA Aptamers Identified R-OH Aptamer:NS5B Gel Shift Binding Assay Experimental Design • Used synthetic DNA library with T7 promoter to synthesize RNA aptamer library • Used immobilized NS5B replicase as target • Identified (4) standard RNA aptamers • Demonstrated inhibition of HCV replication From: Lee et. al. (2013)
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